Probiotic recolonisation therapy

ABSTRACT

The present invention relates to pharmaceutical compositions suitable for the treatment of chronic diseases associated with the presence of abnormal or an abnormal distribution of microflora in the gastrointestinal tract of a mammalian host, which compositions comprise viable non-pathogenic or attenuated pathogenic  Clostridia . The compositions further comprise one or more additional viable non-pathogenic or attenuated pathogenic microorganisms selected from the group consisting of  Bacteroides, Eubacteria, Fusobacteria, Propionibacteria, Lactobacilli, anaerobic cocci, Ruminococcus, E. coli, Gemmiger, Desulfomonas, Peptostreptococcus , and fungi. The present invention also provides pharmaceutical compositions suitable for the treatment of the same chronic diseases comprising viable non-pathogenic or attenuated pathogenic  Escherichia coli , at least one strain of viable non-pathogenic or attenuated pathogenic  Bacteroides  and at least one strain of viable non-pathogenic or attenuated pathogenic microorganism.

CROSS-REFERENCE TO RELATED APPLICATIONS

This U.S. utility patent application is a continuation of U.S. patentapplication Ser. No. 14/793,642, filed Jul. 7, 2015, and is acontinuation of U.S. Ser. No. 14/793,623, filed Jul. 7, 2015, and is acontinuation of U.S. Ser. No. 14/710,481, filed May 12, 2015, and is acontinuation of U.S. Ser. No. 14/710,487, filed May 12, 2015. U.S. Ser.Nos. 14/793,642 and 14/793,623 are each continuations of U.S. Ser. No.14/710,481 and U.S. Ser. No. 14/710,487. U.S. Ser. Nos. 14/710,481 and14/710,487 are each continuations of U.S. Ser. No. 14/270,034, filed May5, 2014, now U.S. Pat. No. 9,050,358, issued Jun. 9, 2015 (which is aContinuation of U.S. Ser. No. 13/910,579, filed Jun. 5, 2013, now U.S.Pat. No. 9,040,036, issued May 26, 2015); and U.S. Ser. No. 13/910,579,filed Jun. 5, 2013, now U.S. Pat. No. 9,040,036, issued May 26, 2015;which is a divisional of U.S. Ser. No. 10/332,986, filed Aug. 4, 2003,now U.S. Pat. No. 8,460,648, issued Jun. 11, 2013; which is a §371national phase of PCT international patent application no.PCT/AU01/00907, having an international filing date of Jul. 25, 2001,which claims benefit of priority to Australian Patent Application SerialNo. PQ 8997, filed Jul. 25, 2000. The aforementioned applications areexpressly incorporated herein by reference in their entirety and for allpurposes.

TECHNICAL FIELD

The present invention relates to pharmaceutical compositions suitablefor the treatment of diseases in mammals, in particular to the treatmentof chronic disorders associated with the presence of abnormal or anabnormal distribution of microflora in the gastrointestinal tract. Theinvention also relates to methods of treating such diseases.

BACKGROUND ART

There are large numbers of patients suffering from gastro-intestinalsymptoms referrable to the lower small bowel and large bowel which todate have eluded explanation. These disorders include irritable bowelsyndrome (IBS) or spastic colon, idiopathic ulcerative colitis, mucouscolitis, collagenous colitis, Crohn's disease, inflammatory boweldisease in general, microscopic colitis, antibiotic-associated colitis,idiopathic or simple constipation, diverticular disease, and AIDSenteropathy. Pathophysiology of these disorders eludes logicalexplanation in spite of decades of research and millions of dollars ofresearch funds. A common underlying factor shared by all these disordersobserved by the present inventor is their onset or aggravation followingsome extraneous invading infection eg travellers diarrhoea. In all thedisorders, a specific causal infection generally cannot be demonstrateddue to our inability to detect infecting agents whose culturalcharacteristics are unknown to medical science.

Circumstantial evidence which suggests that these disorders are“infection-related” includes:

(a) onset following a gastro-intestinal infection which failed tocompletely resolve;

(b) transient improvement with use of certain antibiotics, butrecurrence upon cessation of antibiotics;

(c) transient improvement following orthostatic lavage prior tocolonoscopy and;

(d) transient symptom improvement with use of “colonic” irrigation.

It is impractical to use long-term antibiotic therapy (with itsassociated complications) in such patients since cure is not obtainedwith its use. Furthermore, chronic gut infections with recognised,specific pathogens such as Clostridium difficile, Yersiniaenterocolitica or Campylobacter jejuni/coli are generally not eradicatedwith antibiotics. Some previous attempts have been made to alter theenteric microflora in order to eradicate such chronic infections. Thesemeasures nevertheless indicate that alteration of bacterial flora mayeffect dramatic clinical improvement in conditions characterised bychronic, resistant enterocolitic infection. However there remain manychronic disorders of uncertain aetiology or causation, which areresistant to cure by current therapeutic techniques.

The use of probiotics in the human population has been largely confinedto the inclusion in various foods of live organism of Lactobacilli andBifidobacteria and less frequently Streptococcus faecalis or severalstrains of Escherichia coli. These organisms are thought to promotehealth via immune stimulation and reconstitution of what is presumed tobe normal flora. Such usage stems back to the beliefs generated byMechnikov in the early 1900s. The use of probiotics to treat establishedinfections in the gastrointestinal tract has been lesser but a growingpart of the use of probiotics. Fungal agents such as Saccharomycesboulardii have been used to treat, albeit inefficiently, Clostridiumdifficile infection and Lactobacillus GG has also been used for thispurpose (Floch M. Probiotics and Dietary Fibre. J Clin Gastroenterol1998; 27(2):99-100). Various patents have claimed the use of probioticsfor narrow disease conditions including treatment of Clostridiumdifficile with a combination of Vancomycin and butyric acid bacteria(U.S. Pat. No. 5,266,315), diarrhoea prevention using Lactobacillus(U.S. Pat. No. 5,837,238) or Bifidobacterium (U.S. Pat. No. 5,902,743),Lactobacillus acidophilus to inhibit cryptosporidium (U.S. Pat. No.5,858,356) and mixtures of Lactobacilli and Bifidobacteria in infants toprevent diarrhoea. Enterococcus faecium has been claimed to be useful inalleviating symptoms of Irritable Bowel Syndrome in humans (U.S. Pat.No. 5,902,578) (U.S. Pat. No. 5,728,380) but this has not recognisedClostridium as the underlying agent in this condition. Clostridiumbutyricum as a single agent has been claimed to be a biologicalintestinal antiseptic for treatment of bacterial food poisonings (U.S.Pat. No. 4,892,731), but its use in chronic disease treatment was notcontemplated.

Previous attempts to alter the enteric microflora of a patient haveprescribed the removal of at least a part of the host's existing entericmicroflora, for instance by lavage, prior to substitution withpredetermined desired microflora. This procedure, which was thepreferred embodiment of WO90/01335 has the distinct disadvantages ofcomplicating the treatment and of causing further discomfort to thepatient. This patent also advocated the use of dried, reconstitutedfaeces or a synthetic mixture comprising Bacteroides sp. and Escherichiacoli. It has now been surprisingly found that lavage or other methods ofremoval of at least a part of the host's existing enteric microflora canbe omitted provided a non-pathogenic Clostridium sp. is included withinthe probiotic replacement mixture. Such a replacement mixture has thedual ability of displacing pathogenic bacteria, frequently Clostridialin nature and also establishing a normal environment in which commensalbacteria can establish. Such a treatment permits long-term recovery bothfrom gastrointestinal disorders and from systemic afflictions nothitherto considered to be caused by harmful enteric flora. These arealso called ‘para-infective’ phenomena and can include rheumatological,neurological, regressive, hepatic, and dermatological conditions amongothers.

Autism is a regressive disorder of childhood, affecting boys four timesmore often than girls. It has been observed that the onset of autism isoften preceded by broad spectrum antibiotic use eg for recurrent earinfections. Antibiotic therapy is non-discriminatory in its action andapart from treating the ear infection the microflora of the healthygastrointestinal tract can be severely disrupted by such treatment. Thiscreates an environment where vulnerability to opportunisticmicroorganism colonisation is heightened.

Clostridium tetani is a widely distributed, spore forming anaerobe.Toxigenic strains of Clostridium tetani produce the extremely potenttetanus neurotoxin which is known to enter the central nervous systemfrom the intestinal tract via the vagus nerve (Hensel B et al. NaunynSchmeidebergs Arch Pharmocol 1973; 276:395). Bolte (Med Hypotheses 1998;51:133) has hypothesised that opportunistic infection by Clostridiumtetani may be responsible for the behavioural and medical symptomspresent in a sub-group of individuals diagnosed with autism. Others havealso raised the possibility of clostridia in general as a cause ofdisease (Borriello SP. Clin Infect Dis 1995; Suppl 2:5242).

Sandler et al. (Fourth Int. Symp. Brain-Gut Interactions. 1998; 10: 363)report a trial in which children with delayed onset autism were treatedwith vancomycin over an 8 week period. All children in the trial had hadantecedent broad-spectrum antibiotic exposure, followed by chronicpersistent diarrhoea and then onset of autistic features. Althoughsignificant post-treatment improvement was noted, all childreneventually regressed towards baseline.

It is on the background of these known facts and later the results oftrials of treatment, that the present invention was formulated. Inbrief, it was noted that autistic children (as well as relatedsyndromes) who were referred for treatment of refractory ‘irritablebowel syndrome’ (IBS) viz diarrhoea, flatulence, constipation,distension, abdominal pains etc—responded to treatment of their IBS whentreated with a novel mix of probiotics. However, not only did their IBSimprove dramatically but also their autistic features progressivelyregressed. Even after the initial 2-6 weeks of treatment eye contact wasre-established, repetitive movements were much reduced, and word power(observed vocabulary) expanded—initially 20 words and ultimately >600words at 12 months (estimated), creating ability of the autisticchildren to form long sentences. Continuing improvement was observed tooccur over 12 months of treatment. These observations (to a lesser butdefinite degree at this stage of observations) also applied to thosewith Rett syndrome and children with Attention Deficit/HyperactivityDisorder (ADHD), Attention Deficit Disorder (ADD), and autism variantAsbergers syndrome. The observations strongly suggest that the treatmentof presumed enteric infection/s (eg Clostridial) in these conditions notonly improves the IBS present but also the attendant neurological‘para-infective’ phenomena called collectively autism, Asbergers, Rettsyndrome, ADD or ADHD.

The inclusion within this specification of reference to publisheddocuments is not to be taken to be an admission that any one or more ofthose documents, nor the disclosure of any one or more of thosedocuments, is part of the common general knowledge.

OBJECTS OF THE INVENTION

It is thus an object of the present invention to provide novelpharmaceutical compositions suitable for the treatment of variousdisease states related to the presence of ‘abnormal’ microflora in thegastrointestinal tract. It is a further object of the invention topropose the use of these pharmaceutical compositions in various diseasestates which have not previously been considered to owe their causationto the presence of abnormal flora in the gastrointestinal tract.

DISCLOSURE OF THE INVENTION

The present invention recognises chronic infection/infestation as theunderlying pathological process in a wide range of chronic disorderssuch as irritable bowel syndrome, particularly when characterised bychronic abdominal pain, bloating, or excessive flatulence, together withchronic diarrhoea or alternating constipation/diarrhoea, and also inspastic colon, mucous colitis, collagenous colitis, ulcerative colitis,Crohn's colitis, microscopic colitis, idiopathic inflammatory boweldisease, antibiotic-associated colitis, idiopathic or simpleconstipation, diverticular disease and AIDS enteropathy.

The invention has also been found to relate to other gastrointestinaldisorders of unexplained aetiology such as polyposis coli and colonicpolyps, which may well be influenced by the local bowel microflora.

In addition the present invention also provides a method of treatment ofchronic gastrointestinal infections with specific microorganisms such asClostridium difficile, Yersinia spp, Campylobacter spp, Aeromonas spp,Escherichia coli, Cryptosporidium spp, Amoebae, Blastocystitis homini's,Giardia and even chronic viral infections, and of small bowel bacterialovergrowth.

The present invention furthermore, recognises the close associationbetween the intestine and liver disease, and the intestine and migrainesand chronic fatigue syndrome, and possibly other neurological syndromessuch as, multiple sclerosis, amyotrophic lateral sclerosis, myastheniagravis, Parkinson's disease, Alzheimer's disease, Chronic InflammatoryDemyelinating Polyneuropathy (CIDP), Guillain Barre Syndrome, and otherdegenerative disorders. Hence, it is proposed that a considerableproportion of currently unexplained diseases of the liver and nervoussystem of unknown aetiology may be explicable by the chronic growth ofpathogens within the small/large intestine and the subsequent passage ofantigenic material, pathogenic toxins or biological response modifiers(BRMs) into the portal system (liver damage) or systemic circulationwith antibody formation (neurological conditions). Specifically, suchhepato/biliary system disorders as primary biliary cirrhosis, primarysclerosing cholangitis, fatty liver of unknown aetiology, or cryptogeniccirrhosis, may be secondary to chronic pathogen carrier state in theintestine.

The links between the intestine and joint disease are also recognised.Joint diseases such as rheumatoid arthritis, the non-rheumatoidarthritidies including, ankylosing spondylitis, and Reiter's syndrome,may also be causally related to a chronic intestinal carrier state, asmay other syndromes with an immune mediated component such asglomerulonephritis, haemolytic uraemic syndrome, juvenile diabetesmellitus, Behcet's syndrome, coeliac disease and dermatitisherpetiformis. Similarly, syndromes with an immune complex mediatedcomponent, such as scleroderma, systemic lupus erythematosus, mixedcryoglobulinaemia, polyarteritis, familial Mediterranean fever,amyloidosis, and the various presentations of such syndromes, togetherwith such “idiopathic” states as chronic urticaria, may bemanifestations of variations of immune regulated responses to relatedbowel-origin pathogens chronically shedding their antigen(s), toxins orbiological response modifiers into the circulation. Other chronicconditions such as acne, and chronic idiopathic pseudo-obstructivesyndrome, may well be influenced by similar mechanisms.

For many of these syndromes present therapy offers only palliation ofsymptoms and/or the induction of remission of the disease process butnot cure. The present inventor therefore recognised the need to find acurative therapy for these wide ranging disease processes associatedwith considerable morbidity.

By judicious selection of the microorganisms of the invention it hasbeen surprisingly found by the present inventor that lastingrecolonisation of the gut microflora does not require pretreatment toremove a portion of the host's existing enteric microflora. Thus, byincorporation of Clostridia spp. in the therapy, it has beensurprisingly found that the prior art requirement for removal of atleast a portion of the existing enteric microflora before administrationof the substitute microflora is rendered unnecessary. Without theaddition specifically of Clostridia species, the use of probioticmixtures, eg such as those of bacteroides and Escherichia coli failed tohave the necessary impact on the above-mentioned clinical disorders forthe treatment to be clinically useful. It required a prior purging ofthe gut of its presumably infected and abnormal bowel flora, recolonisation with bacteroides and Escherichia coli—the main componentsof lower intestinal tract, and ongoing feeding of patients with suchbacteria until colonisation was established. The use of Clostridiaappears to be the mainstay of this new therapy and the Clostridia appearto have power of themselves to remove offending bacterial species whichmay be responsible for the underlying condition (presumably pathogenicclostridia—yet to be identified scientifically). Hence, the combinationof non-pathogenic clostridia together with the crucial major colonicbacterial components of bacteroides and Escherichia coli can now be usedas oral therapy to crowd out/destroy/replace and recolonise thedysbiotic flora of patients with various gastrointestinal conditionswhich are caused by abnormal bowel flora. In fact, such a therapybecoming available has_permitted or allowed greater understanding of thepathogenesis of many other conditions which hitherto were thought to becaused by degenerative, inflammatory, or auto immune mechanisms.

Thus according to a first embodiment of the invention there is provideda pharmaceutical composition useful for the treatment and/or prophylaxisof chronic disorders associated with the presence in thegastrointestinal tract of a mammalian host of abnormal or an abnormaldistribution of microflora, which composition comprises viablenon-pathogenic or attenuated pathogenic Clostridia.

Typically the composition includes Clostridia selected from the groupconsisting of Clostridium absonum, Clostridium argentinense, Clostridiumbaratii, Clostridium bifermentans, Clostridium botulinum, Clostridiumbutyricum, Clostridium cadaveris, Clostridium camis, Clostridiumcelatum, Clostridium chauvoei, Clostridium clostridioforme, Clostridiumcochlearium, Clostridium difficile, Clostridium fallax, Clostridiumfelsineum, Clostridium ghonii, Clostridium glycolicum, Clostridiumhaemolyticum, Clostridium hastiforme, Clostridium histolyticum,Clostridium indolis, Clostridium innocuum, Clostridium irregulare,Clostridium limosum, Clostridium malenominatum, Clostridium novyi,Clostridium oroticum, Clostridium paraputrificum, Clostridiumperfringens, Clostridium piliforme, Clostridium putrefaciens,Clostridium putrificum, Clostridium ramosum, Clostridium sardiniense,Clostridium sartagoforme, Clostridium scindens, Clostridium septicum,Clostridium sordellii, Clostridium sphenoides, Clostridium spiroforme,Clostridium sporogenes, Clostridium subterminale, Clostridium symbiosum,Clostridium tedium, Clostridium tetani, Clostridium welchii, Clostridiumvillosum.

In a preferred form the composition further comprises one or moreadditional viable non-pathogenic or attenuated pathogenic microorganismsselected from the group consisting of Bacteroides, Eubacteria,Fusobacteria, Propionibacteria, Lactobacilli, anaerobic cocci,Ruminococcus, Escherichia coli, Gemmiger, Desulfomonas,Peptostreptococcus, species and, more specifically, bacteria selectedfrom Table 1. Preferably fungi are also present such as Monilia.

In a preferred form the composition comprises Clostridia, Bacteroides,Peptostreptococcus, Escherichia coli, Bifidobacterium, andLactobacillus.

In a more preferred form the composition comprises Clostridium innocuum,Clostridium bifermentans, Clostridium butyricum, Bacteroides fragilis,Bacteroides thetaiotaomicron, Bacteroides uniformis, one or more strainsof Escherichia coli, and one or more strains of Lactobacillus.

Alternatively, in a preferred form the composition comprises Clostridiumbifermentans, Clostridium innocuum, and Clostridium butyricum incombination one or more strains of Escherichia coli, one or more strainsof bacteroides and Peptostreptococcus productus.

According to a second embodiment of the invention there is provided apharmaceutical composition useful for the treatment and/or prophylaxisof chronic disorders associated with the presence in thegastrointestinal tract of a mammalian host of abnormal or an abnormaldistribution of microflora, which composition comprises viablenon-pathogenic or attenuated pathogenic Escherichia coli, at least onestrain of viable non-pathogenic or attenuated pathogenic Bacteroides,and at least one other viable non-pathogenic or attenuated pathogenicmicroorganism.

In a preferred form the other viable non-pathogenic or attenuatedpathogenic microorganism is selected from the group consisting ofClostridia, Peptostreptococcus, Bifidobacterium, and Lactobacillus.

Typically the composition of the first or second embodiments of theinvention is derived from disease screened fresh homologous faeces,equivalent freeze-dried and reconstituted faeces or a “synthetic” faecalcomposition. The fresh homologous faeces does not include an antibioticresistant population.

Typically, the composition of the first or second embodiments of theinvention is a synthetic faecal composition.

In a preferred form the synthetic faecal composition comprises apreparation of viable flora which preferably in proportional content,resembles normal healthy human faecal flora which does not includeantibiotic resistant populations. Suitable microorganisms may beselected from the following: Bacteroides, Eubacteria, Fusobacteria,Propionibacteria, Lactobacilli, anaerobic cocci, Ruminococcus,Escherichia coli, Gemmiger, Clostridium, Desulfomonas,Peptostreptococcus, Bifidobacterium, species and, more specifically,bacteria selected from Table 1. Preferably fungi are also present suchas Monilia.

In a preferred form the composition of the first or second embodimentsof the invention comprises a liquid culture.

Preferably, the composition of the first or the second embodiments ofthe present invention is lyophilised, pulverised and powdered. It maythen be infused, dissolved such as in saline, as an enema.

Alternatively the powder may be encapsulated as enteric-coated capsulesfor oral administration. These capsules may take the form ofenteric-coated microcapsules. As a powder it can preferably be providedin a palatable form for reconstitution for drinking or forreconstitution as a food additive. The composition can be provided as apowder for sale in combination with a food or drink. Typically, the foodor drink is a dairy-based product or a soy-based product. The inventiontherefore also includes a food or food supplement containing acomposition according to the first or second embodiment. In a preferredform the food or food supplement contains enteric-coated microcapsulesof the composition of the invention. In a preferred form the food isyogurt.

The powder may be reconstituted also to be infused via naso-duodenalinfusion.

The composition can be combined with other adjuvants such as antacids todampen bacterial inactivation in the stomach, eg Mylanta, Mucaine,Gastrogel. Acid secretion in the stomach could also be pharmacologicallysuppressed using H2-antagonists or proton pump inhibitors. Typically,the H2-antagonist is ranitidine. Typically the proton pump inhibitor isomeprazole.

The composition of the first or second embodiments of the invention istherefore preferably in the form of:

-   -   an enema composition which can be reconstituted with an        appropriate diluent, or    -   enteric-coated capsules, or    -   enteric-coated microcapsules, or    -   powder for reconstitution with an appropriate diluent for        naso-enteric infusion or colonoscopic infusion, or    -   powder for reconstitution with appropriate diluent, flavouring        and gastric acid suppression agent for oral ingestion, or    -   powder for reconstitution with food or drink, or    -   food or food supplement comprising enteric-coated microcapsules        of the composition, powder, jelly, or liquid.

According to a third embodiment of the invention there is provided amethod for the treatment and/or prophylaxis of a chronic disorderassociated with the presence in the gastrointestinal tract of amammalian host of abnormal or an abnormal distribution of microflora,which method comprises administering an effective amount of acomposition according to the first or second embodiment of theinvention.

In its preferred form the treatment should effect a cure of the symptomsof such disorders. The change of flora is preferably as “near-complete”as possible and the flora is replaced by viable organisms which willcrowd out any remaining, original flora.

The method of the present invention is applicable to animals in general,in particular humans and economically significant domestic animals.

In the case of humans, the present invention encompasses methods oftreatment of chronic disorders associated with the presence of abnormalenteric microflora. Such disorders include but are not limited to thoseconditions in the following categories:

-   -   gastro-intestinal disorders including irritable bowel syndrome        or spastic colon, functional bowel disease (FBD), including        constipation predominant FBD, pain predominant FBD, upper        abdominal FBD, non-ulcer dyspepsia (NUD), gastro-oesophageal        reflux, inflammatory bowel disease including Crohn's disease,        ulcerative colitis, indeterminate colitis, collagenous colitis,        microscopic colitis, chronic Clostridium difficile infection,        pseudomembranous colitis, mucous colitis, antibiotic associated        colitis, idiopathic or simple constipation, diverticular        disease, AIDS enteropathy, small bowel bacterial overgrowth,        coeliac disease, polyposis coli, colonic polyps, chronic        idiopathic pseudo obstructive syndrome;    -   chronic gut infections with specific pathogens including        bacteria, viruses, fungi and protozoa;    -   viral gastrointestinal disorders, including viral        gastroenteritis, Norwalk viral gastroenteritis, rotavirus        gastroenteritis, AIDS related gastroenteritis;    -   liver disorders such as primary biliary cirrhosis, primary        sclerosing cholangitis, fatty liver or cryptogenic cirrhosis;    -   rheumatic disorders such as rheumatoid arthritis, non-rheumatoid        arthritidies, non rheumatoid factor positive arthritis,        ankylosing spondylitis, Lyme disease, and Reiter's syndrome;    -   immune mediated disorders such as glomerulonephritis, haemolytic        uraemic syndrome, juvenile diabetes mellitus, mixed        cryoglobulinaemia, polyarteritis, familial Mediterranean fever,        amyloidosis, scleroderma, systemic lupus erythematosus, and        Behcets syndrome;    -   autoimmune disorders including systemic lupus, idiopathic        thrombocytopenic purpura, Sjogren's syndrome, haemolytic uremic        syndrome or scleroderma;    -   neurological syndromes such as chronic fatigue syndrome,        migraine, multiple sclerosis, amyotrophic lateral sclerosis,        myasthenia gravis, Gillain-Barre syndrome, Parkinson's disease,        Alzheimer's disease, Chronic Inflammatory Demyelinating        Polyneuropathy, and other degenerative disorders;    -   psychiatric disorders including chronic depression,        schizophrenia, psychotic disorders, manic depressive illness;    -   regressive disorders including Asbergers syndrome, Rett        syndrome, attention deficit hyperactivity disorder (ADHD), and        attention deficit disorder (ADD);    -   the regressive disorder, autism;    -   sudden infant death syndrome (SIDS), anorexia nervosa;    -   dermatological conditions such as, chronic urticaria, acne,        dermatitis herpetiformis and vasculitic disorders.

The above disorders are all characterised by their response to treatmentwith the method of the present invention.

Typically the change in enteric flora comprises introduction of an arrayof predetermined flora into the gastro-intestinal system, and thus in apreferred form the method of treatment comprises substantiallycompletely displacing pathogenic enteric flora in patients requiringsuch treatment.

Furthermore, in some of these disorders a short course of antibioticsprior to probiotic treatment may be preferred to rid tissue-invasivepathogens originating in the bowel lumen. For example, in Crohn'sdisease, anti-tuberculosis therapy may be required for six to twelveweeks before the bowel is cleared out and the flora content exchangedfor a predetermined flora.

Typically the antibiotic is an anti-Clostridial antibiotic such asvancomycin, rifampicin, and nitroimidazole or chloramphenicol. Typicallythe nitroimidazole is metronidazole.

In a preferred form of the invention, the method of treatment orprophylaxis further includes administration of at least one acidsuppressant prior to administering, or in co-administration with, thecomposition of the invention.

In a preferred form of the invention the method of treatment orprophylaxis further includes nasogastric and/or nasoduodenal washoutprior to administering said composition.

The introduction of the composition into the gastro-intestinal systemcan be effected by enema or per-colonoscope, via intubation of the smallbowel using for example a large bore catheter equipped with distalballoon to effect rapid passage down the jejunum, or via the oral routewith enteric-coated capsules, including enteric-coated microcapsules, orvia the oral route with a supplemented food or drink.

In a preferred form the supplemented food or drink is a dairy-based orsoy-based product. Typically the supplemented food product is yogurt.

According to the method of the invention each dose of the composition isin the range of about 10³ cells to about 10¹³ cells. Preferably eachdose is in the range of about 10⁵ cells to about 10¹¹ cells. Morepreferably each dose is in the range of about 10⁹ cells to about 10¹¹cells. In a preferred form of the invention an initial treatment regimenconsisting of about 10¹⁰ cells per dose is administered about 3 to 6times per day for a period sufficient to stabilise the gut flora.According to the method of the invention the treatment regimen may thencomprise a maintenance dose of about 10¹⁰ cells per day.

Furthermore the present invention also relates to the treatment ofanimals, in particular to the treatment of gastrointestinal disorders ineconomically important domestic animals, such as cattle, sheep, horses,pigs, goats etc. The method of the present invention has been found tobe especially useful in the treatment of the various forms ofnecrotising enterocolitis which can be a major problem in animal stocks.

Obviously in the treatment of animals the appropriate composition ofmicroflora will vary according to the species being treated and theconstituent normal flora known to inhabit the gut. Thus the compositionaccording to the invention would comprise, a preparation of viable florawhich preferably in proportional content, resembles the normal healthyfaecal flora of the species involved. The compositions may be preparedin any of the forms already described and administered accordingly.

Best Method of Performing the Invention

In the practice of the invention a synthetic faecal composition ofpredetermined flora in the form of a liquid or dry powdered culture ofClostridia, Bacteroides, Peptostreptococcus, Escherichia coil,Bifidobacterium, and Lactobacillus, which composition does not includeantibiotic resistant populations, is prepared as a liquid culture.

Typically the method of the invention is applicable to a patientsuffering from a chronic disorder associated with the presence ofabnormal microflora in the gastrointestinal tract such as irritablebowel syndrome.

In the practice of the invention a composition of predetermined flora inthe form of a liquid culture of Clostridia, Bacteroides,Peptostreptococcus, Escherichia coli, Bifidobacterium, and Lactobacillusis ingested by the patient in an amount sufficient to replace andrecolonise the dysbiotic flora of the gastrointestinal tract, andreverse the disease process. Alternatively fresh homologous faecesobtained from a disease screened donor are liquefied and mixed withunprocessed bran. The mixture is then homogenised anaerobically underCO₂ cover and infused into the patient per colonoscope.

Cure or remission of symptoms is then monitored subjectively and byassessment of stool frequency or other appropriate criteria.

Using liquid cultures of Clostridia, Bacteroides, Peptostreptococcus,Escherichia coli, Bifidobacterium, and Lactobacillus the inventor hasachieved total reversal of colitis, irritable bowel syndrome andconstipation.

As indicated in the method of treatment aspect of the invention, apreparatory course of appropriate antibiotics may be used. For example,Septrin for chronic yersiniasis, Metronidazole for ulcerative colitis,anti-TB therapy in Crohn's disease, or Vancomycin in chronic Clostridiumdifficile infestations.

TABLE 1 % of flora ^(b) Organism(s) 11.8(0.90)  Bacteroides fragilis ss.Vulgatus 9.9(0.83) Eubacterium aerofaciens 8.9(0.78) Bacteroidesfragilis ss. Thetaiotaomicron 6.6(0.68) Peptostreptococcus productus II6.0(0.64) Bacteroides fragilis ss. Distasonis 4.4(0.55) Fusobacteriumprausnitzii 3.5(0.49) Coprococcus eutactus 3.0(0.45) Eubacteriumaerofaciens III 2.8(0.44) Peptostreptococcus productus I 2.7(0.43)Ruminococcus bronii 2.6(0.43) Bifidobacterium adolescentis 2.2(0.39)Gemmiger formicilis, Bifidobacterium longum 2.1(0.38) Eubacteriumsiraeum 1.8(0.35) Ruminococcus torques 1.7(0.34) Eubacterium rectaleIII-H 1.6(0.33) Eubacterium rectale IV, Eubacterium eligens 1.5(0.32)Bacteroides eggerthii 1.4(0.31) Clostridium leptum 1.3(0.29) Bacteroidesfragilis ss. A 1.2(0.29) Eubacterium biforme 0.91(0.25)  Bifidobacteriuminfantis 0.84(0.24)  Eubacterium rectale III-F 0.57(0.20)  Coprococcuscomes, Bacteroides capillosus 0.50(0.18)  Ruminococcus albus,Eubacterium formicigenerans, Eubacterium hallii, Eubacterium ventriosumI, Fusobacterium russii 0.43(0.17)  Ruminococcus obeum, Eubacteriumrectale II, Clostridium ramosum I, Lactobacillus leichmanii 0.36(0.16) Ruminococcus cailidus, Butyrivibrio crossotus 0.30(0.14) Acidaminococcus fermentans, Eubacterium ventriosum, Bacteroides fragilisss. fragilis, Bacteroides AR 0.23(0.12)  Coprococcus catus, Eubacteriumhadrum, Eubacterium cylindroides, Eubacterium ruminantium, EubacteriumCH-1, Staphylococcus epidermidis 0.17(0.10)  Peptostreptococcus BL,Eubacterium limosum, Bacteroides praeactus, Bacteroides L, Fusobacteriummortiferum I, Fusobacterium naviforme, Clostridium innocuum, Clostridiumramosum, Propionibacterium acnes, Ruminococcus flavefaciens 0.10(0.08) Ruminococcus AT, Peptococcus AU-1, Eubacterium AG, -AK, -AL, -AL-1, -AN;Bacteroides fragilis ss. ovatus, -ss, d, -ss, f; Bacteroides L-1, L-5;Fusobacterium nucleatum, Fusobacterium mortiferum, Escherichia coli,Streptococcus morbiliorum 0.05(0.05)  Peptococcus magnus, Peptococcus G,-AU-2; Streptococcus intermedius, Ruminococcus lactaris, Ruminococcus COGemmiger X, Coprococcus BH, -CC; Eubacterium tenue, Eubacterium ramulus,Eubacterium AE, -AG-H, -AG- M, -AJ, -BN-1; Bacteroides clostridiiformisss. clostridliformis, Bacteroides coagulans, Bacteroides orails,Bacteroides rumlnicola ss. brevis, -ss. ruminicola, Bacteroidessplanchnlcus, Desuifomonas pigra, Bacteroides L-4, -N-i; FusobacteriumH, Lactobacillus G, Succinivibrio A ^(b) The percentage of the faecalpopulation (the standard deviation of the estimate is given inparentheses).

The invention will now be further described with reference to thefollowing non-limiting examples.

EXAMPLES Formulations

The probiotic therapeutic agents may be prepared in liquid cultureanaerobically or aerobically (depending on bacterium cultured) in pureform. Alternatively the probiotics may be cultured on solid media andscraped into a liquid carrier. The resulting product may be spray-driedinto a powder form and encapsulated or combined with excipients to bedelivered in sachets.

Combinations of Clostridia, Escherichia coli, Bacteroides, andPeptostreptococcus with or without Lactobacilli, Bifidobacteria andEubacteria may be used in varying disorders.

Example No 1 43 Year Old Female

Patient with long standing constipation not responsive to high-dosefibre usage together with prokinetics and standard anti-constipationtreatments, was treated with increasing doses of orally administeredbacterial mix (mixture composition included Clostridium innocuum,bifermentans, butyricum, together with Bacteroides fragilis,thetaiotaomicron and uniformis. Three strains of Escherichia coli werealso included, as was Lactobacillus). This was ingested twice daily inthe first two weeks and then daily thereafter. The patient was not givenany pre-treatment purgative nor any antibiotics. However, she did takeRanitidine (an acid suppressant) three hours prior to ingestion of thebacterial mix. Two weeks after commencing the treatment the patientsconstipation—which would prevent her from defecating for up to fourdays—reversed to increased frequency with reduction of bloating.Initially, gas production increased and there was burbulance andgurgling in the abdomen but after four weeks of treatment the patientwas defecating on a daily basis with no sensation of incomplete emptyingand an almost total absence of bloating. Following the treatment sheremained virtually normal, defecating on a daily basis with 3 monthfollow up.

Example No 2 4½ Year Old Male

Patient with 3 year history of diagnosis of autism associated withIrritable Bowel Syndrome characterised by constipation alternating withdiarrhoea and flatulence, with foul motions, was treated with oraladministration of bacterial mix consisting of Clostridium bifermentans,Clostridium innocuum, and Clostridium butyricum in combination withthree strains of Escherichia coli, three strains of bacteroides andPeptostreptococcus productus. These were ingested following acidsuppression with Ranitidine and were at first taken 3 times daily,reducing to twice daily and then once daily maintenance for eight weeks.The patient's autistic symptoms were reversed quite dramatically withword power increasing from 20 to 200 words (counted by teacher atspecial ‘autistic’ school), he began to sleep through the night, and hisIBS-type symptoms reverted to near-normality with less constipation,less diarrhoea and less foul flatulence. He developed eye contact, wasable to speak sentences up to six words constructed to commands and hebegan to look, to the untrained eye, as a relatively normal child byabout week 10.

Example 3 Male Child, 5½ Years Old

Male child, 5½ years of age with autism symptoms dating back to age ofaround 15 months—but diagnosed significantly later. The patientpresented initially with gastrointestinal symptoms in association withclassical autism—for treatment of the bowel symptoms. Although stooltest did not indicate any specific pathogen the bowel symptoms resembledthose of a chronic infection or adult Irritable Bowel Syndrome (IBS), ieintermittent diarrhoea, constipation, cramping, colicky pain, inabilityto sleep at night, occasional explosive diarrhoea and incontinence. Thepatient was treated with orthostatic lavage using sodium pico-sulfatefollowed by water to produce voluminous diarrhoea and to flush out theenteric contents. He was then given 125 mg Vancomycin three times dailyorally followed by oral re-colonisation with bacteria at a concentrationof 10⁹ through to 10¹⁰, suspended in yoghurt—of strains which includedbacteroides, Escherichia coli, and non pathogenic Clostridia—includingClostridium innocuum, bifermentans and ramosum. The response was quitenoticeable, in the reversal of the abnormal stool function towardsnormality. The patient was also able to sleep through the night withoutany explosive diarrhoea and produced formed stools within five days ofcommencing the bacterial therapy. While the bacteriotherapy wascontinued the bowel symptoms were well controlled. Within 3-4 weeks ofmissing out the treatment for a week or two some of the symptoms wouldbegin to recur. This suggested that the abnormal bacterial flora wassuppressed rather than being cured with this treatment in this patient.The unexpected finding however, was a noticeable and marked reversal ofsymptoms of autism. Whereas previously repetitive movements were presentwith lack of eye contact, eye contact returned fairly rapidly togetherwith cessation of repetitive movement and progressive increase of wordpower from around 20 words to around 600 words by the sixth month oftreatment. The therapy continues now for more than 12 months withsustained reversal of autism and IBS symptoms.

Example 4 Male Child, 7 Years Old

A seven year old male patient was referred for treatment initially ofbowel problems. He had developed autism between age 1 and 2 yearscharacterised by lack of eye contact, repetitive movements, poorlydeveloped cognitive abilities, vocabulary of fewer than 20 words Themarked bowel symptoms were characterised by either constipation or largevoluminous motions, sometimes diarrhoea and explosive stools. Stoolexamination was negative.

The patient was given a pre-treatment of Vancomycin 125 mg twice dailyand at one week he was given an orthostatic lavage consisting ofpicosulfate preparation which flushed out his bowel. He was then giventwice daily oral bacteriotherapy consisting of cultures containingliving probiotics. These included several bacteroides species,Escherichia coli and non-pathogenic Clostridia such as Clostridiumbutyricum, Clostridium bifermentans and Clostridium innocuum. Within twoweeks the bowel symptoms reversed to normal defecation with soft, formedstool—once or twice per day. Constipation disappeared, eye contactreturned over the next six weeks and vocabulary and word use quitedramatically improved, to everyone's surprise. When followed for eightmonths over 600 words could be counted in the vocabulary with sentencesof up to eight words being constructed where previously this was notpossible. Some abstract thinking was noted by teachers at the specialautism school. Parents in particular noted reduced aggression, greaterco-operation, and general increasing ability to develop a more normalrelationship with the child. Repetitive action also disappeared.

Example 5 Male Child, 6 Years Old

A male patient aged 6 was referred to the clinic for treatment ofchronic diarrhoea and at times incontinence. The child had been autisticsince the age of one year and three months. The diagnosis however wasdelayed. He had slow cognitive development and very limited vocabulary.There was virtually absent eye contact and at times violent andexplosive behaviour. The greatest problem with management was that ofcontrol of defecation as the child developed a fascination with thestools which would then be spread over furniture and walls. This broughtsevere pressure upon the family with respect to difficulty withmanagement. Stool test was collected and again was negative for anypathogen. The patient was given Vancomycin 250 mg twice daily for 10days after which a polyethylene glycol orthostatic lavage achieved alarge volume flush of the bowel. He was then given twice daily oralbacteriotherapy in a neutral yogurt as a carrier. Within one week thebowel function returned to virtual normality. However, the behaviouralchanges were just as rapid in reversing again characterised by fairlyrapid reduction in aggressiveness and uncontrollable behaviour, sleepingthrough the night, increased eye contact, and progressively increasedword power. The behaviour of spreading stools also disappeared, more asa behavioural change than learnt phenomenon. The patient was continuedon medications for over a year and progressively improved in allparameters—at times fluctuating in severity.

The invention claimed is:
 1. A method for achieving sustained improvement of one or more autistic symptoms in an autistic subject in need thereof, which method comprises orally administering to said subject an effective amount of a pharmaceutical composition comprising a viable non-pathogenic Clostridium, a viable non-pathogenic Bacteroides, and a viable non-pathogenic Escherichia coli, wherein said method does not require the removal of a portion of said subject's existing enteric microflora via orthostatic lavage prior to said oral administration of said pharmaceutical composition.
 2. The method of claim 1, wherein said sustained improvement lasts for more than 12 months.
 3. The method of claim 1, wherein said method results in continuing improvement over the entire treatment period.
 4. The method of claim 1, wherein said method comprises an initial treatment regimen having a higher dosing frequency followed by a maintenance regimen having a lower dosing frequency.
 5. The method of claim 1, wherein said method comprises an initial treatment regimen having a higher dosage followed by a maintenance regimen having a lower dosage.
 6. The method of claim 1, wherein said subject exhibits one or more symptom improvements selected from the group consisting of reduced repetitive action, sleeping through the night, increased eye contact, and progressively increased word power.
 7. The method of claim 1, wherein said pharmaceutical composition is administered once, twice, or three times daily.
 8. The method of claim 1, wherein said pharmaceutical composition is in a capsule adapted for enteric delivery.
 9. The method of claim 1, wherein said pharmaceutical composition is in an enteric-coated capsule or an enteric-coated microcapsule.
 10. The method of claim 1, wherein said pharmaceutical composition is lyophilized.
 11. The method of claim 1, wherein said pharmaceutical composition comprises no antibiotic resistant populations.
 12. The method of claim 1, wherein said pharmaceutical composition comprises between about 10⁹ and about 10¹¹ viable microbes.
 13. The method of claim 1, wherein said viable non-pathogenic Clostridium comprises a species selected from the group consisting of Clostridium absonum, Clostridium argentinense, Clostridium baratii, Clostridium bifermentans, Clostridium botulinum, Clostridium butyricum, Clostridium cadaveris, Clostridium camis, Clostridium celatum, Clostridium chauvoei, Clostridium clostridioforme, Clostridium cochlearium, Clostridium difficile, Clostridium fallax, Clostridium felsineum, Clostridium ghonii, Clostridium glycolicum, Clostridium haemolyticum, Clostridium hastiforme, Clostridium histolyticum, Clostridium indolis, Clostridium innocuum, Clostridium irregulare, Clostridium limosum, Clostridium malenominatum, Clostridium novyi, Clostridium oroticum, Clostridium paraputrificum, Clostridium perfringens, Clostridium piliforme, Clostridium putrefaciens, Clostridium putrificum, Clostridium ramosum, Clostridium sardiniense, Clostridium sartagoforme, Clostridium scindens, Clostridium septicum, Clostridium sordellii, Clostridium sphenoides, Clostridium spiroforme, Clostridium sporogenes, Clostridium subterminale, Clostridium symbiosum, Clostridium tertium, Clostridium tetani, Clostridium welchii, and Clostridium villosum.
 14. The method of claim 1, wherein said pharmaceutical composition comprises at least two species selected from the group consisting of Clostridium bifermentans, Clostridium innocuum, Clostridium ramosum, and Clostridium butyricum.
 15. The method of claim 1, wherein said viable non-pathogenic Bacteroides comprises a species selected from the group consisting of Bacteroides caccae, Bacteroides coagulans, Bacteroides eggerthii, Bacteroides forsythus, Bacteroides fragilis, Bacteroides gracilis, Bacteroides levil, Bacteroides macacae, Bacteroides merdae, Bacteroides ovatus, Bacteroides pneumosintes, Bacteroides putredinis, Bacteroides pyogenes, Bacteroides stercoris, Bacteroides tectum, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroides ureolyticus, and Bacteroides vulgatus.
 16. The method of claim 1, wherein said viable non-pathogenic Bacteroides comprise a species selected from the group consisting of Bacteroides fragilis, Bacteroides thetaiotaomicron, and Bacteroides uniformis.
 17. The method of claim 1, wherein said effective dose comprises between about 10⁵ and about 10¹¹ viable microbes, between about 10⁷ and about 10⁹ viable microbes, or between about 10⁹ and about 10¹¹ viable microbes.
 18. A method for treating autism in a subject in need thereof, which method comprises orally administering to said subject an effective amount of a pharmaceutical composition comprising a viable non-pathogenic Clostridium, a viable non-pathogenic Bacteroides, and a viable non-pathogenic E. coli, wherein said method does not require the removal of a portion of said subject's existing enteric microflora via orthostatic lavage prior to said oral administration of said pharmaceutical composition.
 19. The method of claim 18, wherein said treatment achieves sustained improvement of one or more autistic symptoms lasting for more than 12 months. 